Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a ...technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen–antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor–ligand interactions.
The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD), Crohn disease (CD), and ulcerative colitis (UC) are only partially ...understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression.
High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11), CD patients (n = 10) and UC patients (n = 10). 33P-radiolabeled cDNA from purified poly(A)+ RNA extracted from biopsies (unpooled) was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome) and CDH11 (cadherin 11, type 2). By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology.
A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.
Background: The spatial gap between cochlear implants (CIs) and the auditory nerve limits frequency selectivity as large populations of spiral ganglion neurons (SGNs) are electrically stimulated ...synchronously. To improve CI performance, a possible strategy is to promote neurite outgrowth toward the CI, thereby allowing a discrete stimulation of small SGN subpopulations. Brain-derived neurotrophic factor (BDNF) is effective to stimulate neurite outgrowth from SGNs. Method: TrkB (tropomyosin receptor kinase B) agonists, BDNF, and five known small-molecule BDNF mimetics were tested for their efficacy in stimulating neurite outgrowth in postnatal SGN explants. To modulate Trk receptor-mediated effects, TrkB and TrkC ligands were scavenged by an excess of recombinant receptor proteins. The pan-Trk inhibitor K252a was used to block Trk receptor actions. Results: THF (7,8,3′-trihydroxyflavone) partly reproduced the BDNF effect in postnatal day 7 (P7) mouse cochlear spiral ganglion explants (SGEs), but failed to show effectiveness in P4 SGEs. During the same postnatal period, spontaneous and BDNF-stimulated neurite outgrowth increased. The increased neurite outgrowth in P7 SGEs was not caused by the TrkB/TrkC ligands, BDNF and neurotrophin-3 (NT-3). Conclusions: The age-dependency of induction of neurite outgrowth in SGEs was very likely dependent on presently unidentified factors and/or molecular mechanisms which may also be decisive for the age-dependent efficacy of the small-molecule TrkB receptor agonist THF.
There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not
previously available. Here we show a glass chip containing 2413 nonredundant ...purified human fusion proteins on a polymer surface,
where densities up to 1600 proteins/cm 2 on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions
under nondenaturing conditions. Such screenings require only 200-μl sample volumes, illustrating their potential for high-throughput
applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity;
and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first
application, we have incubated these protein chips with anti-RGSHis 6 , anti-GAPDH, and anti-HSP90β antibodies. In an initial proof of principle study for the second application, we have screened
serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins
to generate novel diagnostic markers may be possible.
Glomerular hypertension is a major determinant advancing progression to end‐stage renal failure. Podocytes, which are thought to counteract pressure‐mediated capillary expansion, are increasingly ...challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowman's capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell‐matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up‐regulated by stretch (approximately threefold). By reverse transcriptase‐polymer chain reaction, up‐regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate‐salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV‐coated membranes accelerated stretch‐induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch‐adapting molecule in podocytes.
► The yeast–hyphae-dimorphism is a major virulence factor of Candida albicans. ► A reporter assay was optimised to detect hyphal inhibitors in an automated setting. ► A pilot screening of a ...collection of novel compounds was carried out. ► Three new classes of antimycotic components were identified.
Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model.
We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37°C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzodisoxazolones and thiazolo4,5-ebenzoisoxazoles.
In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans.
Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial ...infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.
Prestructured MALDI-MS sample supports have been developed that simplify high-throughput analysis of biomolecules and improve the detection sensitivity. The mass spectrometric sample support is ...coated with a thin layer of hydrophobic Teflon that carries an array of 200-μm gold spots, which provide hydrophilic sample anchors. Each transferred sample droplet contacts one anchor, on top of which, after solvent evaporation, the sample is exclusively deposited due to the strongly water repellent nature of the Teflon surface. The initial matrix concentration is kept low, enabling sample up-concentration by more than 2 orders of magnitudes before crystallization commences. As a result, the detection sensitivity is improved as documented by mass spectra recorded from 100 amol of various peptides, 1 fmol of a DNA 20 mer, and 5 fmol of a 130 bp PCR product. Size and spacing of the hydrophilic anchors are optimized for MALDI-MS performance (sample spot size ≈ laser irradiation spot size), for short analysis times (predetermined sample coordinates), and for high throughput sample preparation (sample anchor array according to the 1536 microtiter plate format).
PDF
